Compstatin analogs with improved activity

ABSTRACT

Compounds comprising peptides and peptidomimetics capable of binding the C3 protein and inhibiting complement activation are disclosed. These compounds display improved complement activation-inhibitory activity as compared with currently available compounds. Isolated nucleic acid molecules encoding the peptides are also disclosed.

Benefit is claimed of U.S. Application No. 60/412,220, filed Sep. 20,2002, the entirety of which is incorporated by reference herein.

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the United StatesGovernment may have rights in the invention described herein, which wasmade in part with funding from the National Institutes of Health, GrantNos. AI 30040 and GM 62134.

FIELD OF THE INVENTION

This invention relates to activation of the complement cascade in thebody. In particular, this invention provides peptides andpeptidomimetics capable of binding the C3 protein and inhibitingcomplement activation.

BACKGROUND OF THE INVENTION

The complement system is the first line of immunological defense againstforeign pathogens. Its activation through the classical, alternative orlectin pathways leads to the generation of anaphylatoxic peptides C3aand C5a and formation of the C5b-9 membrane attack complex. Complementcomponent C3 plays a central role in activation of all three pathways.Activation of C3 by complement pathway C3 convertases and its subsequentattachment to target surface leads to assembly of the membrane attackcomplex and ultimately to damage or lysis of the target cells. C3 isunique in that it possesses a rich architecture that provides amultiplicity of diverse ligand binding sites that are important inimmune surveillance and immune response pathways.

Inappropriate activation of complement may lead to host cell damage.Complement is implicated in several disease states, including variousautoimmune diseases, and has been found to contribute to other clinicalconditions such as adult respiratory syndrome, heart attack, rejectionfollowing xenotransplantation and burn injuries. Complement-mediatedtissue injury has also been found to result from bioincompatibilitysituations such as those encountered in patients undergoing dialysis orcardiopulmonary bypass.

Complement-mediated tissue injuries are directly mediated by themembrane attack complex, and indirectly by the generation of C3a andC5a. These peptides induce damage through their effects on neutrophilsand mast cells. In vivo, regulation of complement at the C3 and C5activation steps is provided by both plasma and membrane proteins. Theplasma protein inhibitors are factor H and C4-binding protein, and theregulatory membrane proteins located on cell surfaces are complementreceptors 1 (CR1), decay-accelerating factor (DAF), and membranecofactor protein (MCP). These proteins inhibit the C3 and C5 convertases(multi-subunit proteases), by promoting dissociation of the multisubunitcomplexes and/or by inactivating the complexes through proteolysis(catalyzed by factor I). Several pharmacological agents that regulate ormodulate complement activity have been identified by in vitro assay, butmost have been shown in vivo to be of low activity or toxic.

To date, there are no inhibitors of complement activation used in theclinic, though certain candidates for clinical use exist, specifically,a recombinant form of complement receptor 1 known as soluble complementreceptor 1 (sCR1) and a humanized monoclonal anti-C5 antibody(5G1.1-scFv). Both of these substance's have been shown to suppresscomplement ctivation in in vivo animal models (Kalli et al., SpringerSemin. Immunopathol. 15: 417-431, 1994; Wang et al., Proc. Natl. Acad.Sci. USA 93: 8563-8568, 1996). However, each substance possesses thedisadvantage of being large molecular weight proteins (240 kDa and26,000 kDa, respectively) that are difficult to manufacture and must beadministered by infusion. Accordingly, recent research has emphasizedthe development of smaller active agents that are easier to deliver,more stable and less costly to manufacture.

U.S. Pat. No. 6,319,897 to Lambris et al. describes the use of aphage-displayed combinatorial random peptide library to identify a27-residue peptide that binds to C3 and inhibits complement activation.This peptide was truncated to a 13-residue cyclic segment thatmaintained complete activity, which is referred to in the art asCompstatin. Compstatin inhibits the cleavage of C3 to C3a and C3b by C3convertase. Compstatin has been tested in a series of in vitro, in vivo,ex vivo, and in vivo/ex vivo interface experiments, and has beendemonstrated to: (1) inhibit complement activation in human serum (Sahuet al., J. Immunol. 157: 884-891, 1996); (2) inhibitheparin/protamine-induced complement activation in primates withoutsignificant side effects (Soulika et al., Clin. Immunol. 96: 212-221,2000); (3) prolong the lifetime of a porcine-to-human xenograft perfusedwith human blood (Fiane et al., Transplant. Proc. 31: 934-935, 1999a;Fiane et al., Xenotransplantation 6: 52-65, 1999b; Fiane et al.,Transplant. Proc. 32: 899-900, 2000); (4) inhibit complement activationin models of cardio-pulmonary bypass, plasmapheresis, and dialysisextra-corporeal circuits (Nilsson et al., Blood 92: 1661-1667, 1998);and (5) possess low toxicity (Furlong et al., Immunopharmacology 48:199-212, 2000).

Compstatin is a peptide comprising the sequence ICVVQDWGHHRCT-NH₂ (SEQID NO:1), where Cys2 and Cys12 form a disulfide bridge. Itsthree-dimensional structure was determined using homonuclear 2D NMRspectroscopy in combination with two separate experimentally restrainedcomputational methodologies. The first methodology involved distancegeometry, molecular dynamics, and simulated annealing (Morikis et al.,Protein Science 7: 619-627, 1998) and the second methodology involvedglobal optimization (Klepeis et al., J. Computational Chemistry, 20:1344-1370, 1999). The structure of Compstatin revealed a molecularsurface that comprises of a polar patch and a non-polar patch. The polarpart includes a Type I β-turn and the non-polar patch includes thedisulfide bridge. In addition, a series of analogs with alaninereplacements (an alanine scan) was synthesized and tested for activity,revealing that the four residues of the β-turn and the disulfide bridgewith the surrounding hydrophobic cluster are essential for inhibitoryactivity (Morikis et al., 1998, supra).

Using a complement activity assay comprising measuring alternativepathway-mediated erythrocyte lysis, the IC₅₀ of Compstatin has beenmeasured as 12 μM. Certain of the analogs previously tested havedemonstrated activity equivalent to, or slightly greater than,Compstatin. The development of Compstatin analogs or mimetics withgreater activity would constitute a significant advance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs and mimetics of thecomplement-inhibiting peptide, Compstatin (ICVVQDWGHHRCT-NH₂; SEQ IDNO:1), which have improved complement-inhibiting activity as compared toCompstatin.

In one aspect, the invention features a compound that inhibitscomplement activation, which comprises a peptide having a sequence:

(SEQ ID NO: 15) Xaa1-Cys-Val-Xaa2-Gln-Asp-Trp-Gly-Xaa3-His-Arg-Cys-Xaa4;

-   wherein:-   Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide    comprising Gly-Ile;-   Xaa2 is Trp or a peptidic or non-peptidic analog of Trp;-   Xaa3 is His, Ala, Phe or Trp;-   Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, or a tripeptide comprising    Thr-Ala-Asn, wherein a carboxy terminal —OH of any of the L-Thr,    D-Thr, Ile, Val, Gly or Asn optionally is replaced by —NH₂; and the    two Cys residues are joined by a disulfide bond.

In certain embodiments, Xaa1 is acetylated, and typically is Ac-Ile. Inanother embodiment, Xaa3 is Ala. In other embodiments, Xaa2 is an analogof Trp comprising a substituted or unsubstituted aromatic ringcomponent, preferably comprising a bicyclic ring, (e.g., indole,napthyl) or two rings (e.g., dibenzoyl). In exemplary embodiments, theanalog of Tip is 2-napthylalanine, 1-naphthylalanine, 2-indanylglycinecarboxylic acid, dihydrotryptophan or benzoylphenylalanine.

In a particular embodiment, Xaa1 is Ac-Ile, Xaa2 is Trp or an analog ofTrp comprising a substituted or unsubstituted indole, naphthyl ordibenzoyl component, Xaa3 is Ala and Xaa4 is L-threonine or D-threonine.Exemplary sequences are selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

In another particular embodiment, Xaa1 is a dipeptide Gly-Ile, and Xaa 4is a tripeptide Thr-Ala-Asn. An exemplary embodiment is a peptide havingSEQ ID NO:14.

Another aspect of the invention features a compound that inhibitscomplement activation, comprising a non-peptide or partial peptidemimetic of the peptide described above, wherein one or more of theresidues or analogs is replaced by a compound that enables retained orenhanced complement-activation inhibiting activity.

These compounds are of practical utility for any purpose for whichCompstatin itself is utilized, as known in the art.

Another aspect of the invention features an isolated nucleic acidmolecule encoding one or more peptides that inhibits complementactivation, wherein the peptide comprises a sequence:Xaa1-Cys-Val-Xaa2-Gln-Asp-Trp-Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO:15);

-   wherein:-   Xaa1 is Ile, Val, Leu, or a dipeptide comprising Gly-Ile;-   Xaa2 is Trp;-   Xaa3 is His, Ala, Phe or Trp; and-   Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, or a tripeptide comprising    Thr-Ala-Asn; wherein the two Cys residues are joined by a disulfide    bond.

The isolated nucleic acid molecule typically encodes a peptide whereinXaa3 is Ala. In an exemplary embodiment, the isolated nucleic acidmolecule encodes a peptide comprising SEQ ID NO:14. In anotherembodiment, the nucleic acid encodes a concatemer of two or more of apeptide comprising SEQ ID NO:14, wherein the encoded concatemer iscleavable by hydrazine to form a multiplicity of peptides comprising SEQID NO:14.

Expression vectors comprising any of the aforementioned isolated nucleicacid molecules are featured in another aspect of the invention, alongwith cells comprising the expression vectors, which may be bacterial,fungal, insect, plant or mammalian cells. Peptides encoded by theseisolated nucleic acid molecules are useful for any purpose for whichCompstatin is useful.

Various features and advantages of the present invention will beunderstood by reference to the detailed description, drawings andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of complement activation by Compstatin (SEQ ID NO:1)and Ac-4W-9A-13dT-OH (SEQ ID NO:6). X axis is peptide concentration(μM), Y axis is inhibition of complement activation as measured by theassay described in Example 2; negative control is a linear peptide(“linear”), comprising Compstatin modified with alanine replacingcysteine at positions 2 and 12.

FIG. 2. Inhibition of complement activation by Compstatin (SEQ ID NO:1),Ac-4(2Nal)-9A (SEQ ID NO:7), Ac-4(2Nal)-9A-OH (SEQ ID NO:8) andAc-4(1Nal)-9A-OH (SEQ ID NO:9). X axis is peptide concentration (μM), Yaxis is inhibition of complement activation as measured by the assaydescribed in Example 2; negative control is a linear peptide (“linear”),comprising Compstatin modified with alanine replacing cysteine atpositions 2 and 12.

FIG. 3. Inhibition of complement activation by Compstatin (SEQ ID NO:1)and Ac-4(Ig1)-9A-OH (SEQ ID NO:11). X axis is peptide concentration(μM), Y axis is inhibition of complement activation as measured by theassay described in Example 2; negative control is a linear peptide(“linear”), comprising Compstatin modified with alanine replacingcysteine at positions 2 and 12.

FIG. 4. Inhibition of complement activation by Compstatin (SEQ ID NO:1)and Ac-4(Igl)-9A (SEQ ID NO:10). X axis is peptide concentration (μM), Yaxis is inhibition of complement activation as measured by the assaydescribed in Example 2; negative control is a linear peptide (“linear”),comprising Compstatin modified with alanine replacing cysteine atpositions 2 and 12.

FIG. 5. Inhibition of complement activation by Compstatin (SEQ ID No:1)and Ac-4(Dht)-9A-OH (SEQ ID NO:12). X axis is peptide concentration(μM), Y axis is inhibition of complement activation as measured by theassay described in Example 2; negative control is a linear peptide(“linear”), comprising Compstatin modified with alanine replacingcysteine at positions 2 and 12.

FIG. 6. Inhibition of complement activation by Compstatin (SEQ ID NO:1)and +G-4W-9A-15N-OH (SEQ ID NO:14). X axis is peptide concentration(μM), Y axis is inhibition of complement activation as measured by theassay described in Example 2; negative control is a linear peptide(“linear”), comprising Compstatin modified with alanine replacingcysteine at positions 2 and 12.

FIG. 7. Inhibition of complement activation by Compstatin (SEQ ID NO:1)and Ac-4(Bpa)-9A-OH (SEQ ID NO:13). X axis is peptide concentration(μM), Y axis is inhibition of complement activation as measured by theassay described in Example 2; negative control is a linear peptide(“linear”), comprising Compstatin modified with alanine replacingcysteine at positions 2 and 12.

FIG. 8. Inhibition of complement activation by Compstatin (SEQ ID NO:1),Ac-Compstatin (SEQ ID NO:2), Ac-4W-9A (SEQ ID NO:5) and Ac-4W-9A-OH (SEQID NO:4). X axis is peptide concentration (μM), Y axis is inhibition ofcomplement activation as measured by the assay described in Example 2;negative control is a linear peptide (“linear”), comprising Compstatinmodified with alanine replacing cysteine at positions 2 and 12.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings.

The terms “pharmaceutically active” and “biologically active” refer tothe ability of the compounds of the invention to bind C3 or fragmentsthereof and inhibit complement activation. This biological activity maybe measured by one or more of several art-recognized assays, asdescribed in greater detail herein.

As used herein, “alkyl” refers to a saturated straight, branched, orcyclic hydrocarbon having from about 1 to about 10 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 7 carbon atoms beingpreferred. Alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl,adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

As used herein, “halo” refers to F, Cl, Br or I.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and have from about 6 to about 20 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred. Aralkyl groups can be optionally substituted. Non-limitingexamples include, for example, benzyl, naphthylmethyl, diphenylmethyl,triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(=O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen. Exemplary substituents include, forexample, halo, alkyl, cycloalkyl, aralkyl, aryl, sulthydryl, hydroxyl(—OH), alkoxyl, cyano (—CN), carboxyl (—COOH), —C(═O)O-alkyl,aminocarbonyl (—C(—O)NH₂), —N-substituted aminocarbonyl (—C(═O)NHR″),CF₃, CF₂CF₃, and the like. In relation to the aforementionedsubstituents, each moiety R″ can be, independently, any of H, alkyl,cycloalkyl, aryl, or aralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

100421 In accordance with the present invention, information about thebiological and physico-chemical characteristics of Compstatin have beenemployed to design Compstatin analogs with significantly improvedactivity compared to the parent Compstatin peptide. In preferredembodiments, the analogs have at least 5-fold greater activity than doesCompstatin, preferably using the assay described in Example 2. Morepreferably, the analogs have 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or50-fold greater activity, and even more preferably, 60-, 70- 80-fold orgreater activity than does Compstatin, preferably utilizing the assaydescribed in Example 2.

Compstatin analogs synthesized in accordance with other approaches havebeen shown to possess somewhat improved activity as compared with theparent peptide, i.e., up to six-fold (Klepeis, et al. 2003, JACS 125:8422-8423). The analogs produced in accordance with the presentinvention possess even greater activity either the parent peptide oranalogs thereof produced to date, as demonstrated by in vitro assays asshown in the figures and in Table 1 below.

Table 1 shows amino acid sequence and complement inhibitory activitiesof Compstatin and selected analogs with significantly improved activity.The selected analogs are referred to by specific modifications ofdesignated positions (1-13) as compared to the parent peptide,Compstatin.

TABLE 1 SEQ ID Activity over Peptide Sequence NO: Compstatin CompstatinH-ICVVQDWGHHRCT-CONH2 1 * Ac-Compstatin Ac-ICVVQDWGHHRGT-CONH2 2  3 ×more Ac-4Y,9A Ac-ICV Y QDWG A HRCT-CONH2 3 19 × more Ac-4W,9A-OH Ac-ICVW QDWG A HRCT-COOH 4 25 × more Ac-4W,9A Ac-ICV W QDWG A HRCT-CONH2 555 × more Ac-4W,9A13dT-OH Ac-ICV W QDWG A HRCdT-COOH 6 55 × moreAc-4(2-Nal), 9A Ac-ICV(2-Nal)QDWG A HRCT-CONH2 7 66 × moreAc-4(2-Nal),9A-OH Ac-ICV(2-Nal)QDWG A HRCT-COOH 8 39 × moreAc-4(1-Nal),9A-OH Ac-ICV(1-Nal)QDWG A HRCT-COOH 9 23 × more Ac-4Igl,9AAc-ICVIglQDWG A HRCT-CONH2 10 55 × more Ac-4Igl,9A-OH Ac-ICVIglQDWG AHRCT-COOH 11 55 × more Ac-4Dht,9A-OH Ac-ICVDhtQDWG A HRGT-COOH 12  9 ×more Ac-4(Bpa),9A-OH Ac-ICV(Bpa)QDWG A HRCT-COOH 13 55 × more+G,4W,9A +AN-OH H- G ICV W QDWG A HRCTA N -COOH 14 38 × more dT =D-threonine 2-Nal = 2-napthylalanine 1-Nal = 1-napthylalanine Igl = 2indanylglycine carboxylic acid Dht = dihydrotryptophan Bpa =benzoylphenylalanine

Modifications at the N-terminus. Acetylation of the N-terminus typicallyincreases the complement-inhibiting activity of Compstatin and itsanalogs, as can be seen specifically by comparing SEQ ID NO: 1 with SEQID NO:2. Accordingly, N-acetylation of the peptide is one preferredembodiment of the invention, of particular utility when the peptides areprepared synthetically. However, it is sometimes of advantage to preparethe peptides by expression of a peptide-encoding nucleic acid moleculein a prokaryotic or eukaryotic expression system, or by in vitrotranscription and translation. For these embodiments, thenaturally-occurring N-terminus is utilized. One example of a Compstatinanalog suitable for expression in vitro or in vivo is that of SEQ IDNO:14, wherein the acetyl group is replaced by unmodified glycine at theN-terminus. SEQ ID NO:14, which additionally comprises Trp at positionX4, Ala at position X9, and a C-terminal extension of Ala-Asn atpositions X14 and X15, is 38-fold more active than Compstatin in thecomplement inhibition assay described herein.

Modification within the peptide. Using computational methods that therank low lying energy sequences, it was previously determined that Tyrand Val were the most likely candidates at position 4 to supportstability and activity of the peptide (Klepeis, et al., 2003, supra). Inview of that determination, the present discovery that Trp at position4, especially combined with Ala at position 9, yields many-fold greateractivity than that of the parent peptide, is unexpected (for example,compare activities of SEQ ID NOS: 4, 5 and 6 with those of SEQ ID NOS: 2and 3. Trp might be expected to contribute to the hydrophobic clusterinvolving residues at positions 1, 2, 3, 4, 12, and 13; however, itsbulky side-chain mandates against the ability of a peptide comprisingTrp to maintain its active conformation. Nonetheless, in accordance withthe invention, Trp at position 4 of the peptide has been empiricallydetermined to contribute significantly to the activity of the peptide.

Without intending to be limited by any particular mechanism of action,Trp at position 4 of the peptide may enhance activity by virtue of acation-n interaction between the Trp aromatic side chain and cationicelements of the region of C3 with which the peptide interacts. It hasbeen established that cation-π interaction, which is the electrostaticattraction between a cation and the negative electrostatic potentialassociated with the face of a simple π system, can contributesubstantially to the binding of ligands to a broad range of proteinclasses (for a review, see Zacharias & Dougherty 2002, TIBS 23:281-287).

Accordingly, modifications of Trp at position 4 (e.g., altering thestructure of the side chain according to methods well known in the art),or substitutions of Trp analogs that maintain or enhance theaforementioned cation-π interaction, are contemplated in the presentinvention to produce analogs with even greater activity. For example,peptides comprising the tryptophan analogs 2-napthylalanine (SEQ ID NOS:7, 8), 1-napthylalanine (SEQ ID NO: 9), 2-indanylglycine carboxylic acid(SEQ ID NOS: 10, 11) or dihydrotryptophan (SEQ ID NO: 12) at position 4were all found to possess increased complement-inhibitory activity,ranging from 9-fold to 66-fold greater than Compstatin. In addition, apeptide comprising the phenylalanine analog, benzoylphenylalanine, atposition 4 (SEQ ID NO: 13) possessed 55-fold greater activity that didCompstatin. It is believed that the planar two-ring compositions ofthese indole, napthyl or dibenzoyl compounds enhances the π interactionafforded by the analog at position 4, thereby increasing the activity ofthe peptide. Accordingly, Trp analogs comprising two or more aromaticrings are preferred for use in the present invention. Such analogs arewell known in the art and include, but are not limited to the analogsexemplified herein, as well as unsubstituted or alternativelysubstituted derivatives thereof. Examples of suitable analogs may befound by reference to the following publications, and many others:Beene, et al. (2002) Biochemistry 41: 10262-10269 (describing, interalia, singly- and multiply-halogenated Trp analogs); Babitzke & Yanofsky(1995) J. Biol. Chem. 270: 12452-12456 (describing, inter alia,methylated and halogenated Trp and other Trp and indole analogs), andU.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097,4,576,750 and4,299,838.

Modifications at the carboxy terminus. Peptides produced by syntheticmethods are commonly modified at the carboxy terminus to comprise anamide instead of an acid; this common modification can be seen in Table1 in Compstatin (SEQ ID NO:1) and several analogs. Indeed, in someinstances, it has been determined that the terminal amide-containingpeptides possess greater activity than do the terminal acid-containingpeptides (compare, for example, SEQ ID NOS: 5 and 7 with SEQ ID NOS: 4and 8, respectively). Accordingly, one preferred embodiment of theinvention utilizes the C-terminal amide modification. However, somecircumstances favor the use of a the acid at the C-terminus. Suchcircumstances include, but are not limited to solubility considerationsand the expression of the peptides in vitro or in vivo frompeptide-encoding nucleic acid molecules.

The carboxy-terminal residue of Compstatin is threonine. In someembodiments of the present invention, the C-terminal threonine isreplaced by one or more naturally-occurring amino acids or analogs. Forexample, the peptide having SEQ ID NO:6 comprises D-threonine instead ofL-threonine, and further possesses a COOH group at the C-terminus. Thispeptide shows activity equal to that of peptide SEQ ID NO:5, comprisingL-threonine and CONH₂ at the C-terminus. Further, Ile has beensubstituted for Thr at position 13, to obtain a peptide with 21-foldgreater activity than that of Compstatin. In addition, the peptide ofSEQ ID NO:14, which comprises a C-terminal dipeptide extension ofAla-Asn, along with a COOH at the C-terminus and a non-acetylatedN-terminus, demonstrates 38-fold greater activity than does Compstatin.It is also suitable for production via a prokaryotic or eukaryoticexpression system, as described in greater detail below.

Another peptide that shows an increase in activity as compared withCompstatin comprises modifications in the N-terminal residue and withinthe peptide. This peptide comprises Ac-Leu at position 1, Trp atposition 9 and Gly at position 13, but is unmodified at position 4.

The Compstatin analogs of the present invention may be prepared byvarious synthetic methods of peptide synthesis via condensation of oneor more amino acid residues, in accordance with conventional peptidesynthesis methods. Preferably, peptides are synthesized according tostandard solid-phase methodologies, such as may be performed on anApplied Biosystems Model 431A peptide synthesizer (Applied Biosystems,Foster City, Calif.), according to manufacturer's instructions. Othermethods of synthesizing peptides or peptidomimetics, either by solidphase methodologies or in liquid phase, are well known to those skilledin the art. During the course of peptide synthesis, branched chain aminoand carboxyl groups may be protected/deprotected as needed, usingcommonly-known protecting groups. An example of a preferred peptidesynthetic method is set forth in Example 1. Modification utilizingalternative protecting groups for peptides and peptide derivatives willbe apparent to those of skill in the art.

Alternatively, certain peptides of the invention may be produced byexpression in a suitable procaryotic or eucaryotic system. For example,a DNA construct may be inserted into a plasmid vector adapted forexpression in a bacterial cell (such as E. coli) or a yeast cell (suchas Saccharomyces cerevisiae), or into a baculovirus vector forexpression in an insect cell or a viral vector for expression in amammalian cell. Such vectors comprise the regulatory elements necessaryfor expression of the DNA in the host cell, positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The peptide of SEQ ID NO:14, and others similarly designed, isparticularly preferred for production by expression of a nucleic acidmolecule in vitro or in vivo. A DNA construct encoding a concatemer ofSEQ ID NO:14 (e.g., 2 or more of SEQ ID NO:14; the upper limit beingdependent on the expression system utilized) may be introduced into anin vivo expression system. After the concatemer is produced, cleavagebetween the C-terminal Asn and the following N-terminal G isaccomplished by exposure of the polypeptide to hydrazine.

The peptides produced by gene expression in a recombinant procaryotic oreucyarotic system may be purified according to methods known in the art.In a preferred embodiment, a commercially available expression/secretionsystem can be used, whereby the recombinant peptide is expressed andthereafter secreted from the host cell, to be easily purified from thesurrounding medium.

The structure of Compstatin is known in the art, and the, structures ofthe foregoing analogs are determined by similar means. Once a particulardesired conformation of a short peptide has been ascertained, methodsfor designing a peptide or peptidomimetic to fit that conformation arewell known in the art. See, e.g., G. R. Marshall (1993), Tetrahedron,49: 3547-3558; Hruby and Nikiforovich (1991), in Molecular Conformationand Biological Interactions, P. Balaram & S. Ramasehan, eds., IndianAcad. of Sci., Bangalore, PP. 429-455). Of particular relevance to thepresent invention, the design of peptide analogs may be further refinedby considering the contribution of various side chains of amino acidresidues, as discussed above (i.e., for the effect of functional groupsor for steric considerations).

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce C3-binding, complement-inhibiting compounds through the use ofeither naturally-occurring amino acids, amino acid derivatives, analogsor non-amino acid molecules capable of being joined to form theappropriate backbone conformation. A non-peptide analog, or an analogcomprising peptide and non-peptide components, is sometimes referred toherein as a “peptidomimetic” or “isosteric mimetic,” to designatesubstitutions or derivations of the peptides of the invention, whichpossess the same backbone conformational features and/or otherfunctionalities, so as to be sufficiently similar to the exemplifiedpeptides to inhibit complement activation.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art (see, e.g., Zhao et al. (1995), NatureStructural Biology 2: 1131-1137; Beely, N. (1994), Trends inBiotechnology 12: 213-216; Hruby, V. J. (1993), Biopolymers 33:1073-1082). Assuming rotational constraints similar to those of aminoacid residues within a peptide, analogs comprising non-amino acidmoieties may be analyzed, and their conformational motifs verified, bymeans of the Ramachandran plot (see Hruby & Nikiforovich, supra), amongother known techniques.

The complement activation-inhibiting activity of Compstatin analogs andpeptidomimetics may be tested by a variety of assays known in the art.In a preferred embodiment, the assay described in Example 2 is utilized.A non-exhaustive list of other assays is set forth in U.S. Pat. No.6,319,897, including, but not limited to, (1) peptide binding to C3 andC3 fragments; (2) various hemolytic assays; (3) measurement of C3convertase-mediated cleavage of C3; and (4) measurement of Factor Bcleavage by Factor D.

The peptides and peptidomimetics described herein are of practicalutility for any purpose for which Compstatin itself is utilized, asknown in the art. Such uses include, but are not limited to: (1)inhibiting complement activation in the serum of a patient (human oranimal); (2) inhibiting complement activation that occurs during use ofartificial organs or implants (e.g., by coating or otherwise treatingthe artificial organ or implant with a peptide of the invention); (3)inhibiting complement activation that occurs during extracorporealshunting of physiological fluids (blood, urine) (e.g., by coating thetubing through which the fluids are shunted with a peptide of theinvention); and (4) in screening of small molecule libraries to identifyother inhibitors of compstatin activation (e.g., liquid- or solid-phasehigh-throughput assays designed to measure the ability of a testcompound to compete with a Compstatin analog for binding with C3 or a C3fragment).

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 Peptide Synthesis

Peptide synthesis and purification was performed as described by Sahu etal., 1996, supra, and Sahu et al., 2000, supra. Briefly, peptides weresynthesized in an Applied Biosystem peptide synthesizer (model 431A)using Fmoc amide resin and standard side chain protecting groups.Peptides were cleaved from the resin by incubation for 3 hours at 22° C.with a solvent mixture containing 5% phenol, 5% thioanisole, 5% water,2.5% ethanedithiol, and 82.5% trifluoroacetic acid (TFA). The reactionmixture was filtered through a flitted funnel, precipitated with coldether, dissolved in 50% acetonitrile containing 0.1% TFA, andlyophilized. The crude peptides obtained after cleavage were dissolvedin 10% acetonitrile containing 0.1% TFA and purified using a reversephase C-18 column (Waters, Milford, Mass.). Disulfide oxidation wasachieved by an on-resin cyclization method using the reagent Thallium(III) trifluoroacetate. This method eliminates the dilute solutionoxidation steps and subsequent time-consuming concentration throughlyophilization steps prior to reverse-phase HPLC. Using this method, themultimer formation was nonexistent and a high level (˜90%) of fullydeprotected, oxidized or cyclized material was obtained. The identityand purity of all peptides were confirmed by laser desorption massspectroscopy and HPLC.

EXAMPLE 2 Complement Inhibition Assays

Inhibitory activity of Compstatin and its analogs on the complementsystem was determined by measuring their effect on the activation of thecomplement system by immunocomplexes. Complement activation inhibitionwas assessed by measuring the inhibition of C3 fixation toovalbumin-anti-ovalbumin complexes in normal human plasma. Microtiterwells were coated with 50 μl of ovalbumin (10 mg/ml) for 2 hr at 25° C.(overnight at 4° C.). The wells were saturated with 200 μl of 10 mg/mlBSA for 1 hr at 25° C. and then a rabbit anti-ovalbumin antibody wasadded to form an immunocomplex by which complement can be activated.Thirty microliters of peptides at various concentrations were addeddirectly to each well followed by 30 μl of a 1:80 dilution of humanplasma. After 30 min incubation, bound C3b/iC3b was detected using agoat anti-human C3 HRP-conjugated antibody. Color was developed byadding ABTS peroxidase substrate and optical density measured at 405 nm.The concentration of the peptide causing 50% inhibition of C3b/iC3bdeposition was taken as the IC₅₀ and used to compare the activities ofvarious peptides.

All scientific articles, patents and other publications cited herein areincorporated by reference in their entireties. The present invention isnot limited to the embodiments described and exemplified above, but iscapable of variation and modification within the scope of the appendedclaims.

1. A compound that inhibits complement activation, which comprises apeptide having a sequence: (SEQ ID NO: 15)Xaa1-Cys-Val-Xaa2-Gln-Asp-Trp-Gly-Xaa3-His-Arg- Cys-Xaa4;

wherein: Xaa1 is Ile, Val, Leu, Ac-le, Ac-Val, Ac-Leu or a dipeptidecomprising Gly-Ile; Xaa2 is Trp or a peptidic or non-peptidic analog ofTrp; Xaa3 is His, Ala, Phe or Trp; Xaa4 is L-Thr, D-Thr, Ile, Val, Gly,or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal —OHof any of the L-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replacedby —NH2; and the two Cys residues are joined by a disulfide bond.
 2. Thecompound of claim 1, wherein Xaa1 is Ac-Ile.
 3. The compound of claim 1,wherein Xaa3 is Ala.
 4. The compound of claim 1, wherein Xaa2 is ananalog of Trp comprising a substituted or unsubstituted bicyclicaromatic ring component or two or more substituted or unsubstitutedmonocyclic aromatic ring components.
 5. The compound of claim 4, whereinthe analog of Trp is selected from the group consisting of2-napthylalanine, 1-naphthylalanine, 2-indanylglycine carboxylic acid,dihythotryptophan and benzoylphenylalanine.
 6. The compound of claim 1,wherein Xaa1 is Ac-Ile, Xaa2 is Trp or an analog of Trp comprising asubstituted or unsubstituted indole, naphthyl or dibenzoyl component,Xaa3 is Ala and Xaa4 is L-threonine or D-threonine.
 7. The compound ofclaim 6, having a sequence selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.
 8. Thecompound of claim 1, wherein Xaa1 is a dipeptide Gly-Ile, and Xaa 4 is atripeptide Thr-Ala-Asn.
 9. The compound of claim 8, comprising a peptidehaving SEQ ID NO:14.
 10. A compound that inhibits complement activation,comprising a non-peptide or partial peptide mimetic of the compound ofclaim 1, wherein the compound binds C3 and inhibits complementactivation with at least five-fold greater activity than does a peptidecomprising SEQ ID NO:1, under equivalent assay conditions.
 11. Anisolated nucleic acid molecule encoding one or more peptides thatinhibits complement activation, wherein the peptide comprises asequence: (SEQ ID NO: 15)Xaa1-Cys-Val-Xaa2-Gln-Asp-Trp-Gly-Xaa3-His-Arg- Cys-Xaa4;

wherein: Xaa1 is Ile, Val, Leu, or a dipeptide comprising Gly-Ile; Xaa2is Trp; Xaa3 is His, Ala, Phe or Trp; and Xaa4 is L-Thr, D-Thr, Ile,Val, Gly, or a tripeptide comprising Thr-Ala-Asn; wherein the two Cysresidues are joined by a disulfide bond.
 12. The isolated nucleic acidmolecule of claim 11, encoding a peptide wherein Xaa3 is Ala.
 13. Theisolated nucleic acid molecule of claim 12, encoding a peptidecomprising SEQ ID NO:14.
 14. The isolated nucleic acid molecule of claim13, encoding a concatemer of two or more of a peptide comprising SEQ IDNO:14, wherein the encoded concatemer is cleavable by hydrazine to forma multiplicity of peptides comprising SEQ ID NO:14.
 15. An expressionvector comprising the isolated nucleic acid molecule of claim
 11. 16. Acell comprising the expression vector of claim
 15. 17. The cell of claim16, which is a bacterial, fungal, plant, insect or mammalian cell.